Tuesday 6th May 2014
This week began on Tuesday, as Monday was a bank holiday. I started the week off by incubating my newly made plugs in 0.1 x wash buffer for 1 hour. This was then removed and replaced with 1 x NotI buffer for another hour with gentle agitation. This was again removed and replaced with 1 x NotI buffer and NotI restriction enzyme, incubating overnight at 37ºC.
Wednesday 7th May 2014
I began today by collecting the bacterial plugs from incubation, removing the buffer and enzyme then incubating the plugs in 1 x wash buffer for an hour with agitation. This was then removed and replaced with 0.5 x TAE buffer. While they were incubating in TAE I prepared and poured a 1% agarose gel.
This gel was then loaded with the plugs, an appropriate PFGE ladder and my new Lambda ladder before running overnight using the following settings:
- 20 hours
- 1% agarose gel
- 0.5x TAE
- 40-100 switch intervals per second
- 6 V/cm
- 30 minute stain, 10 minute destain
I decided to run this gel using the same settings as my last most successful PFGE gel, in order to visulise whether this new batch of plugs contained a more appropriate amount of bacterial DNA alongside my new lambda ladder.
Thursday 8th May 2014
Once my PFGE gel had finished I stained it for 30 minutes, destaining for 10 minutes before viewing under UV. The resulting gel was fairly disappointing, while the new ladder appeared blurred my original PFGE ladder was very faint and the Escherichia coli bacterial plugs slightly on the dark side. The settings I have optimised for my original PFGE ladder and Escherichia coli bacterial plugs did not give appropriate banding patterns for my new Lambda ladder.
Unfortunately I don’t have enough time to run another gel again this week to confirm that my new ladder is not running correctly, so I will have to wait until my Bradford sample collection is complete.
Finally I collected my autoclaved Duran flasks, extendable rod and gloves to take home so that we could head straight off to Bradford in the morning.
Friday 9th May 2014
I began today fairly early, meeting my research supervisor and driving up to Bradford for water collection. After around 2 hours we reached Bradford, and headed towards Riddlesdon to explore the stretch of the river Aire into which the nearby waste water treatment plant (WWTP) disposed it’s processed effluent. As neither of us had been here to collect water before we spent around an hour and half finding the best way down to the river, taking multiple samples down stream from the WWTP and finally collecting a control sample from upstream. It was really good to get to explore a new river collection site, especially with my supervisor, as it opens up the potential for students to come back to this site for further research work.
Once we were happy with all the samples taken we headed back to Lincoln and into the lab to start water processing. I began by filtering my collected water through filter membranes and applying these to MLSA plates, using the same method as before. These plates were then incubated overnight at 44ºC.
Saturday 10th May 2014
In order to get all my Bradford water samples processed and stocked I decided to work over the weekend. I removed the incubated MLSA plates and inoculated single colonies from the plates into separate LB broths. These were then incubated overnight at 44ºC.
Sunday 11th May 2014
Today started with removing the incubated LB broths and inoculating each into lactose peptone water (LPW) and tryptone water (TW) before incubating overnight at 44ºC. 10μl of each overnight sample was then spread plated onto mueller hinton agar, allowed to absorb before applying with antibiotic susceptibility discs. These plates were also incubated overnight at 44ºC.
I then inoculated each overnight sample onto an individual nutrient agar slope, storing at room temperature to allow slow growth and access to single colonies. Finally, I glycerol stocked all the samples to ensure I have reserves of all isolated Bradford Escherichia coli samples. I pipetted 500μl of each overnight LB broth culture into separate tubes before adding 500μl of 10% glycerol LB broth. These were then carefully labelled and stored in the freezer.
Now I have all my Bradford water samples processed, Escherichia coli isolated and stored I will be moving onto plasmid extraction using the QIAcube. This will again allow me to see which Escherichia coli isolates carry plasmids, and should therefore be investigated through PCR for the presence of specific genes (Bla genes and CTX-M).
If I have time I would also like to carry on finalising PFGE settings with my new Lambda ladder. Once finished, hopefully I will be able to begin producing individual bacterial plugs for each Escherichia coli sample that demonstrated banding patterns when ran on standard gel electrophoresis. As I have only made 10 plugs at one time from only 3 samples I will need to set aside plenty of time to make plugs from all my Lincoln and Bradford samples!
A Year in the Lab…